It is well recognized that all amino acids exist in one of two isomer forms, known as the L-form and its mirror image, the D-form. The aromatic amino acid phenylalanine, for example, has two optically active enantiomers, L-phenylalanine and D-phenylalanine. In its major application, a component of aspartame the popular low calorie sweetener, it is preferred to separate the two isomers or convert a racemic mixture of the two to obtain one of the enantiomers that is relatively free of contamination by the other. Aspartame itself is a dipeptide comprised of two amino acids, aspartic acid and phenylalanine. It has been known for quite some time that the sweetening property of the dipeptide is dependent upon the stereochemistry of these individual amino acids.
Since both of these amino acids can exist in either the D (dextro) or L (levo) form, it has been determined that L-aspartyl-L-phenylalanine esters are sweet while the corresponding D-D, D-L and L-D isomers are not. Combinations of the isomers which contain the L-L dipeptide, DL-aspartyl-L-phenylalanine, L-aspartyl DL-phenylalanine and DL-aspartyl-DL-phenylalanine are sweet, but only half as sweet since the racemate contains 1/2 of the L-L moiety. Hence, in the manufacture of aspartame, it is most desirable to have a starting material of phenylalanine that is comprised of purely the L-isomer form.
Phenylalanine is conventionally made either through chemical synthesis, enzymatic synthesis or fermentation. Most of these methods result in a racemic mixture of the D and L isomers which must either be separated or resolved, to yield a pure L-phenylalanine fraction. Ideally, a method or process whereby the D isomers could be converted to the L isomer would not only do away with the need for the additional and costly separation or resolution steps, but it would also increase the final yield of the desired product.
Biocatalytic processes resulting in the formation of a specific isomer are known in the art. For example, L-phenylalanine can be produced via enzymatic synthesis from cinnamic acid and ammonia using the enzyme phenylalanine-ammonia lyase. L-alanine may also be produced in a pure fraction by the enzymatic decarboxylation of L-aspartic acid. See U.S. Pat. Nos. 3,458,400 and 3,463,704. Wood et al., U.S. Pat. No. 4,600,692 also utilized transaminases in the preparation of L-phenylalanine and oxaloacetate from phenylpyruvic acid, pyridoxal-5-phosphate and an amine donor such as L-glutamate or L-aspartate in a reversible reaction.
Of particular interest is Rozzell, J., U.S. Pat. No. 4,518,692. A process is disclosed therein whereby L-amino acids are produced by transamination. The process comprises reacting an alpha-keto acid or a racemic mixture of an amino acid with L-aspartic acid in the presence of a transaminase enzyme to produce an alpha amino acid corresponding to the alpha keto-acid and oxaloacetate which must be subsequently decarboxylated. The transamination reaction requires a multi-enzyme system comprising the addition of oxaloacetate decarboxylase, D-amino acid oxidase and pyridoxal-5-phosphate which are not needed in the process of the present invention. D-amino acid oxidase is a mammalian enzyme extracted from hog kidney. A byproduct of this reaction is hydrogen peroxide which has been shown to inactivate enzymes and can cause decomposition of 2-ketoacids. The enzyme is also expensive and thereby increases the cost of the overall process.
Also of interest to the background of the present invention is U.S. Pat. No. 4,745,061 to Aretz et al. which discloses a D-amino acid transaminase that has been isolated from Bacillus licheniformis. The enzyme is shown to be useful in the preparation of D-amino acids from .DELTA. keto acids and is suitable for resolving racemates of D,L amino acids by converting the D-amino acid to its corresponding .alpha.-keto acid which is then separated. The recovery of pure L-amino acid would thus require costly purification and separation steps not necessary in the practice of the present invention.
It is an object of the present invention to provide a process for the production of L-amino acids using a transaminase that is stereospecific in the synthesis of L-amino acids. More specifically, it is an object of the present invention to provide a process for the production of optically pure L-phenylalanine from a D, L racemic mixture using a transaminase from a culture without the required addition of aspartate, D-amino acid oxidase, oxaloacetate decarboxylase or catalase.